The document discusses the estimation of starch using the anthrone reagent method, highlighting starch's role as a vital polysaccharide and its hydrolysis into simple sugars. It outlines a procedure for extracting starch from samples and detailing colorimetric measurement processes, emphasizing the necessary chemical reactions and concentrations. Additionally, it provides materials needed, calculated nutritional values, and references for further reading.

1. Jasmine Juliet .R
Biochemistry
Agricultural College &
Research Institute,
Madurai.

2. Estimation of starch by anthrone reagent
 Starch is an important polysaccharide.
 It is the storage form of carbohydrate in plants abundantly
found in roots, tubers, stems, fruits and cereals.
 Starch, which is composed of several glucose molecules, is a
mixture of two types of components namely amylose and
amylopectin.
 Starch is hydrolysed into simple sugars by dilute acids and the
quantity of simple sugars is measured colorimetrically.

3. Starch analysis by Anthrone method
 The anthrone method is one of the colorimetric methods used to
determine starch concentration or total sugar content in food.
 Under acidic conditions, sugars react with anthrone reagents to
produce a blue-greenish color.
 Heating needs to be conducted when reacting samples with sulfuric
acid and reagents with the aim of accelerating the reaction rate, then
cooled and measured its absorbance at 630 nm.
 Sugars reacted with sulfuric acid, it acts as a strong oxidizing agent.

4. Principle
The sample is treated with 80% alcohol to
remove sugars and then starch is extracted with
perchloric acid.
In hot acidic medium starch is hydrolysed to
glucose and dehydrated to hydroxymethyl
furfural.
This compound forms a green coloured product
with anthrone.

5. Estimation of starch by anthrone reagent

7. Blank colour is yellow. Standard colour intensity increases
with standard concentration increases.

8. Materials
Anthrone: Dissolve 200mg anthrone in 100mL of
ice-cold 95% sulphuric acid
80% ethanol
52% perchloric acid
Standard Glucose:
Stock – 100mg in 100mL water.
Working Standard – 10ml of stock diluted to 100mL
with water.

9. Procedure
 Homogenize 0.1 to 0.5g of the sample in hot 80% ethanol to remove
sugars. Centrifuge and retain the residue. Wash the residue repeatedly
with hot 80% ethanol till the washing do not give color with anthrone
reagent. Dry the residue well over a water bath.
 To the residue add 5.0mL of water and 6.5mL of 52% perchloric acid.
 Extract at 0°C for 20min. Centrifuge and save the supernatant.
 Repeat the extraction using fresh perchloric acid. Centrifuge and pool
the supernatant and make up to 100mL.

10. Procedure
Pipette out 0.1 or 0.2mL of the supernatant and
make up the volume to 1mL with water.
Prepare the standards by taking 0.2, 0.4, 0.6, 0.8 and
1mL in each tube with water.
Add 4mL of anthrone reagent to each tube.
Heat for eight minutes in a boiling water bath.
Cool rapidly and read the intensity of green to dark
green color at 630nm.

12. S.No
Vol. of
w.std
(ml)
Conc. of
w.std (μg)
Vol. of
water
(ml)
Vol. of
anthrone
reagent (ml)
Keep
in
boiling
water
bath
for
8
minutes
OD at
630nm
Blank
S1
S2
S3
S4
S5
T1
T2
Observation and Calculation

13. Calculation
Find out the glucose content in the sample using the
standard graph.
Multiply the value by a factor 0.9 to arrive at the starch
content.
Calculate the starch percentage by using the formula,
Starch content = Glucose content x 0.9.

14. References
Hodge, J E and Hofreiter, B T (1962) In:
Methods in Carbohydrate Chemistry (eds
Whistler, R L and Be Miller, J N) Academic
Press New York.
Thayumanavan, B and Sadasivam, S (1984) Qual
Plant Foods Hum Nutr 34 253.

15. Video link
• https://www.youtube.com/watch?v=pXAf1QO
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• https://www.youtube.com/watch?v=C6IloM6
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